Microorganisms commonly encounter threatening changes in their environments. These changes include depletion of nutrients, shifts in pH, temperature and pressure, and sharing habitats with invading organisms. A commonly encountered stress of animal-associated bacteria and one of importance to a study of infectious diseases is that induced through the transfer of cultures from their propagation media into different media. This stress is especially pronounced when the bacteria are transferred to a media of neutral pH with altered or reduced levels of nutrients, such as when exogenous bacteria enter an animal or when endogenous bacteria penetrate sterile zones and tissues from their non-sterile habitats. During growth, bacteria form H+ which accumulates in both the cell and the media.
When encountering normal and naturally-occurring stresses, bacteria release products which the inventor has named stress response factors, (SRFs). These SRFs include polymers of nucleic acids and their partial and complete hydrolysates including a mixture of about a dozen oligoribonucleotides (ORN) ranging from 1-30 nucleotides, which are resistant to further hydrolysis by RNAse.
The present inventor has found that a series of mild stresses, mimicking those commonly experienced when bacterial environments change, induces the release of these oligoribonucleotides. Their release does not kill or injure the bacteria from which they were released, but rather assists them in establishing a colony in a new environment by increasing their rate of mutation and their rate of growth.
These oligoribonucleotides are released when bacteria enter the pH neutral environment of animals, such as the mouth, nose, oropharyngeal cavity, urethra and vagina.
Through co-evolution in a shared habitat, the immune systems of animals and humans have adapted a protective response to the appearance of these ORN in anticipation of a microbial invasion. This immune response to the released ORN precedes its detection of bacteria entering the pH neutral body fluids or attempting to invade horizontally onto sterile tissue, (e.g. from the nose to the sinus, from vagina to uterus). Encountering neutral pH will induce the release of these ORN, which will serve to alert the host to a potential penetration onto a sterile area or into sterile tissue. This immune response follows the stimulation of embedded sentinel cells, e.g. in oral pharyngeal tissue as well as the circulating sentry cells monocytes and macrophages, and B- and T-cells of the immune system.
Specifically, the macrophage has adapted a preemptory reaction to the presence of the oligoribonucleotides (ORN) that prepares the immune system to defend the host against infection. For example, when ingested bacteria encounter neutral pH or are overcrowded by the presence of growing pathogens, they will release readily absorbable, non-toxic ORN which activate tissue macrophages to release Interleukin-1, IL-1, Interleukin-6, IL-6, and Tumor Necrosis Factor, alpha, TNFα which stimulate other cells of the immune system. After being highly activated, exposed macrophages down-regulate the surface receptors, CD-14 and CD-16, thereby desensitizing the cell from over-activation by the subsequent interaction with bacterial toxins if infection occurs.
The present inventor has found that the oligomeric fraction having a molecular weight <10 kDa and, in particular, between 500 and 3,000 Da are readily absorbed, are non-toxic, and both activate and modulate the immune system. The products less than 10 kDa in size are non-toxic when fed or injected and contain further a group of compounds of oligomeric size, i.e. 0.5 to 3 kDa that activate and modulate macrophages. Macrophages are activated to release cytokines at levels deemed helpful to combating infections and are also down-modulated to prevent their over-activation with its subsequent release of host-threatening levels of cytokines and becoming overly cytotoxic resulting in the perforation of organs.
As sentry cells, macrophages circulate in the blood and lymph as well as reside in specialized endothelial tissues and organs. They are among the host's first lines of defense, releasing interleukin signals, destroying microbes and stimulating other immune cells to destroy diseased cells of the host. Approximately twenty different interleukins can be released modifying, amplifying, restricting and dampening messages as the system is stimulated. Thus, the macrophage's signal is key to initiating and enforcing the appropriate immune response. In an infection, bacterial endotoxin (lipopolysaccharide, LPS), binds to the TOLL-like receptor, TLR-4 and the CD-16 surface molecules on macrophages stimulating them and inducing the release of yet higher levels and combinations of IL-1, IL-6 and TNF. These signals, in turn, induce fever, fatigue, cardiovascular hypotension, renal failure and can dictate the death of the host in “septic shock”.
By down-regulating the numbers of CD-14 and CD-16 receptors on the surface of the macrophage, the oligomers released by high levels of stressed bacteria help to ensure that the macrophage does not become over-activated by interleukins thereby creating a system which may go awry killing the host.
Monocytes also respond to the presence of released bacterial ORN by escaping apoptosis and maturing into macrophages.
Thirteen different species of animal-associated bacteria have been found to release oligoribonucleotides <10 kDa when stressed. However, the distribution of polymer:oligomer:monomer is not equal amongst these species. The levels of ORN accumulated and released are related to the rate of growth of the bacteria. Therefore, not all strains of bacteria, even of the same species, release levels of oligomers sufficient to protect animals against a subsequent bacterial invasion. Feral colonies of bacteria yield more oligoribonucleotides (ORN) when initially stressed than non-feral or laboratory strains. However, inducing a stress upon a laboratory strain before re-propagation will stimulate its growth rate and accumulation of ORN, comparable to that occurring when a feral strain was stressed.
The fraction >10 kDa is toxic when injected into mice, producing a ruffled fur coat, huddling and diarrhea. However, the fraction <10 kDa activates and modulates macrophages, is non-toxic when fed or injected into mice and protects them against a subsequent lethal challenge of injected endotoxin.
The present inventor has found that the ORN <10 kDa are a rich new source of natural, normally-occurring, co-evolutionarily evolved immune modulators that can be safely used to protect animals and humans from infections and the over-stimulation of their immune system. In addition, this fraction contains compounds that can be used to adjust the expression of individual surface receptors on macrophages to re-center a dysfunctional immune system.
Furthermore, in vitro and in vivo testing indicates the potential role of ORN <10 kDa as adjuvants by stimulating the production of antibodies. Human B-cells were stimulated in cell culture and mice demonstrated increased B-cell activity when an experimental vaccine against melanoma was injected with ORN<10 kDa in a trial.
An unusual feature of the ORN<10 kDa that possess immune stimulating capacity is their resistance to hydrolysis by RNase (product R-7003 purchased from Sigma Chemical Co.) when used as directed by the manufacturer (incubating 750 μL of ORNs with 50 μg of RNase at 37° C. for 3 hr). The pattern of the ORN<10 kDa on Sephadex G-10 was the same before and after RNase treatment showing no further reduction in molecular size.
The resistance of ORN<10 kDa to RNase suggests that the ORN have unusual structures. Some may have substitutions on their bases and ribose (e.g., methyl and other groups) or may have nucleotide strands held together by complementary hydrogen bonding with itself. These unusual structural conditions leading to resistance to RNase are believed to be responsible for their unique stimulation and modulation of the immune system.
Through co-habitation with bacteria, the immune system has evolved to recognize the bacterial origin of these ORN. Besides the unusual structures that prohibited further hydrolysis by RNase, the inventor believes that the immune system recognizes as “bacterial” certain base sequences in the ORN<10 kDa. Their importance to bacteria is evidenced by how well conserved they are across genera. Their ubiquity, consistency and conservation over many genera of bacteria have permitted the immune system to invest a significant response to their sudden appearance.
The <10 kDa ORN collected from heat-killed bacteria stimulate monocytes in cell culture but do not protect mice from endotoxic death. Therefore pasteurized products might be erroneously considered effective if demonstrated in a reductionistic assay similar to the one previously described. (Mangan D F, Welch G R, Wahl S M, 1991, Lipopolysaccharide, tumor necrosis factor-α, and IL-1B prevent programmed cell death (apoptosis) in human peripheral blood monocytes, J Immunol 146:1541-6).
The discovery of the release of immune-activating and modulating ORN <10 kDa has broad implications to improving the immune response through both diet and pharmaceutical preparations for humans and animals. Dairy products like milk, cheese, and yogurts contain viable bacteria, which, when transferred to the nutrient deprived environment of pH neutral body fluids, such as the mouth, release ORN. More effective health outcomes could be gained from consuming such products if they were formulated to release biologically significant amounts of ORN or by extending the dwell-time of the bacteria or ORN in the mouth and throat or by fortifying pasteurized products by adding back the viable bacteria lost during pasteurization or by the addition of ORN possessing greater stimulating powers.
Numerous patents teach the healthful benefits of administering viable bacteria to humans and animals either orally or parenterally to provide immune stimulation. Their mode of action is purported to be due to 1) The aggressive nature of a specific strain of bacteria to eliminate resident pathogens, 2) The release of antibiotics or other chemicals, which act to mitigate the efforts of pathogens, or 3) The release of vitamins or enzymes that are believed to improve the health of the immune system. The prior art also recognizes the use of cytokines to stimulate certain immune responses. However cytokines are toxic and can induce lethal septic shock. None of the prior art teaches that harmless bacteria release specific ORN to which the immune system has adapted an alert response. The present inventor believes that ORN are the molecules responsible for the healthful benefits derived from consuming probiotics or fermented dairy products. Such benefits can be obtained by administering either bacteria containing ORN or the purified ORN themselves released by bacteria.
U.S. Pat. No. 4,975,467 teaches methods by which synthetic compounds can be used to inhibit the release of IL-1 thereby alleviating the induction of its pathophysiologic conditions. U.S. Pat. No. 5,055,447 provides methods and compositions for the prevention of septic shock by administering growth factor-β. This patent teaches the use of administering a signal compound to intercept or modify existing signals. U.S. Pat. Nos. 5,041,427 and 5,158,939 teach the use of a non-toxic LPS from R. spaeroides, ATCC 17023 to desensitize macrophages to toxic LPS. Since R. spaeroides has an unusual lipid A structure, it may not be effective as a desensitizing agent. U.S. Pat. No. 5,157,039 supports the clinical importance for controlling IL-1 release by macrophages by teaching the use of two non-natural quinolinol compounds, which appear to be non-selective in IL inhibition. U.S. Pat. No. 5,840,318 teaches that bacteria exposed to a neutral pH buffer for 10-16 hours release compounds <10 kDa in size having a UV maximum absorption at 254 nm (A-254). Feeding the <10 kDa sizes activates and modulates an animal's immune system, thereby protecting it against a subsequent lethal challenge of endotoxin. The accumulation of A-254 absorbing compounds in bacteria is associated with the slowing or termination of cell growth. Further, U.S. Pat. No. 6,589,771 B1 teaches that bacteria enter dormancy during their stationary phase by accumulating solutes that increase the cytoplasmic osmolality and H+ which decrease the intracellular pH of the cell. Putting dormant cells in buffers with osmolalities and hydrogen ion (H+) concentrations lower than that of the cell will force the diffusion of solutes and H+ thereby inducing the activation of the cell. Rather than a single 10-16 hour wash to induce the release of the compounds that have a maximum of UV absorption at 254 nm, one can employ a series of 20-minute washings in a buffer of pH 7 and of an osmolality lower than the cell's followed by an adjustment period of approximately 72 hours in the final wash.
Livestock are routinely fed silage, a product containing high levels of viable and harmless Lactobacillus plantarum. When ingested and chewed as cud, the silage bacteria release immune-activating ORN. The proper selection of harmless bacteria that ferment silage and grains and also release biologically significant levels of ORNs will help improve the health of livestock.
Direct-fed microbials and probiotics are harmless bacteria which are grown in a rich media, concentrated, dried and fed to animals either as a powder for top-dressing or in gel forms for oral inoculation. On occasion some of these products have been shown to provide a health benefit to the animal in combating infections relating to shipping and weaning. However, analyses of commercial products indicate a low concentration of ORNs as well as their inadequate release when transferred to saliva or to a nutrient-reduced environment. This inventor has determined that the generally accepted methods for preparing probiotics do not allow for the accumulation or retention of significant levels of ORN. It is believed that uncontrolled unit operations during production may occasionally and randomly produce a finished product containing adequate levels of ORN-releasing viable bacteria. These uncontrolled production parameters explain the occasional effectiveness reported for some probiotics. This invention puts forth the production and storage parameters, which yield a bacterial preparation that permits the administration of a stable, probiotic of known bacterial dose or one containing sterile ORN for livestock and poultry. Currently, viability of probiotics is believed necessary for effectiveness. However, this invention allows for the distribution of a sterile, stable product that can be stored without refrigeration and would provide a known dose of a measurable chemical.
The present inventor has found that the extrusion of ORN from the cell is forced by transferring bacteria which have accumulated ORN into environments of a lower molality and lower H+ concentration than that of the bacterial cell, e.g. saliva, body fluids and physiologic saline. Extrusion can be induced by subjecting the bacterial populations to repeated dilutions or washings, as many as 6, in pH neutral buffers of sufficient molarity to maintain a neutral pH in the presence of the efflux of H+ that accompany ORN. Further, the present inventor has discovered doses of ORN that are also effective in rescuing monocytes from apoptosis and, in addition, rescue animals from the lethality of endotoxemia. Moreover, lab tests have been developed which aid in the selection and screening of bacteria, which release the more potent ORN and determining appropriate doses.
ORN are the products of the destruction of ribosomal RNA, which occurs concomitantly with the slowing of bacterial growth. The number of ribosomes is known to be directly proportional to the rate of growth of bacteria. Fast growing populations are known to possess 70,000 ribosomes per bacterial cell while cells in slow-growing population have as few as 2000 ribosomes. (Neidardt, F. C., Ingraham, J. L. and Schaechter, M., in The Physiology of the Bacterial Cell, Sinauer Assoc., Sunderland, Mass., 1990, p 430). As exponential growth slows, ribosomal RNA is hydrolyzed, creating very acidic oligoribonucleotides, which accumulate in the cytoplasm and in the broth. With accumulation of ORNs by the cytoplasm, the lag period extends into days and even weeks.
The hydrolysis of ribosomal RNA requires conditions of neutral pH from about 6 to about 9 but preferably from about 6 to about 8. However, acidic conditions (a pH of or less than 6) are required for the formation and stabilization of the ORN:DNA complex. The first of this two-step reaction is accomplished during the growth of bacteria in neutral broths. At about the mid-exponential growth point (5-8 hrs), H+ begin to be released along with ORNs from the hydrolysis of RNA.
In the manufacture of probiotics the culture broth of bacteria is artificially maintained at neutral pH in order to prevent the slowing of growth and thereby maximizing production of the bacteria for sale. Sodium hydroxide is commonly added to resist the culture's natural tendency towards a lower pH. Short production times and large cell masses are the goals of probiotic manufacturers and are achieved by not allowing the pH of the media to become acidic.
However, under these common practices, ORN may be formed but are not retained in the bacteria. According to the generally-accepted practices, the probiotic bacteria are then concentrated by centrifugation or filtration at neutral pH. Under these pH conditions, the ORN cannot bind the genomic DNA, and are lost to the supernatant, which is flushed down the drain.
The ORN that are most effective in boosting an immune response are those between 0.5 and 10 kDa. They are a new rich source of natural, normally-occurring, co-evolutionarily evolved immune modulators that can be safely used to protect animals and humans from infections and over-stimulation of their immune system. In Pending U.S. patent application Ser. No. 09/883,550 ORNs, ranging from 0.5 to 10. kDa, have been shown to activate and modulate macrophages by adjusting the expression of individual surface receptors on macrophages, thereby re-centering a dysfunctional immune system. Furthermore, human cell culture and in vivo animal testing indicate the potential role of ORN<10 kDa as adjuvants by stimulating the production of antibodies. Results from stimulating human B-cells in cell culture and in mice demonstrate increased B-cell activity when an experimental vaccine against melanoma was injected with ORN<10 kDa. Furthermore, in Pending U.S. patent application Ser. No. 09/883,550 in vitro testing indicates the ORN potential role as adjuvants by stimulating the release of IL-12. See U.S. Pat. No. 5,840,318. Studies with Balb/C mice have revealed that the consumption or injection of sterile preparations of ORNs in this range are non-toxic to the mice and provide the mice protection against a subsequent lethal challenge of injected endotoxins. See U.S. Pat. No. 5,840,318.
Because of the ORN potential as immune modulators, the discovery of the release of immune-activating and modulating factors has broad implications to improving the immune response through diets and pharmaceutical preparations for humans and animals. Numerous patents teach the healthful benefits of administering specific viable bacteria to humans and animals either orally or parenterally to provide local immune stimulation. For example, direct-fed microbials and probiotics, harmless bacteria which are grown in a rich media, concentrated, and dried, may be fed to animals either as a powder for top-dressing or in gel forms for oral inoculation. See U.S. Pat. Nos. 6,797,266; 6,461,607; and 5,401,501. These products are intended to provide a health benefit to the animal in combating infections relating to shipping and weaning.
However, many products marketed as containing probiotics are perceived as over-hyped. J Appl Bacteriol. 1989 May; 66(5):365-78. Clinical testing of probiotic-containing products has generated inconsistent and equivocal results regarding the products' claims. J. Nut. 2000; 130:384S-390S, Nut. Rev. 2003 March; 61(3):91-9. In short, many probiotics claims are unsubstantiated or simply fail to demonstrate any health benefit. The inventor believes that ORN are the molecular mode of action of probiotics and are lost during the presently accepted general methods of producing probiotics. While the prior art recognizes the importance of modulating the immune system, it does not teach the parameters of production to provide a significant level of ORNs which are safe, natural, normally occurring biological chemicals derived from co-evolution, which are effective when taken orally or parenterally.
To date, thirteen different species of animal-associated bacteria have been found to release ORN<10 kDa when stressed. See U.S. Pat. No. 5,840,318. However, the distribution of polymer:oligomer:monomer is not equal amongst these species. See U.S. Pat. No. 5,840,318. Therefore, not all strains of bacteria, even of the same species, release levels of ORN<10 kDa sufficient to protect animals against a subsequent bacterial invasion. Therefore, for these and other reasons, there is a need for the present invention.
It is an object of the present invention to provide a method for the accumulation of ORN in bacteria.
It is an object of the present invention to provide a method for the retention of ORN in bacteria.
It is an object of the present invention to provide a method for preventing the loss of ORN during bacterial production or storage.
It is an object of the present invention to provide an isolated bacterium comprising ORN less than 10,000 Daltons in molecular size.
It is an object of the present invention to provide isolated ORN that are less than 10,000 Daltons in molecular size.
It is an object of the present invention to provide a method of preparing an immune-enhancing animal feed or animal drinking water comprising ORN.
It is an object of the present invention to provide a method of preparing an immune-enhancing animal feed or animal drinking water comprising bacteria having ORN.
It is an object of the present invention to provide a method of enhancing the immune system of an animal or its resistance to infection.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention, which follows hereafter.